U.S. patent application number 10/007389 was filed with the patent office on 2003-09-04 for methods for the analysis of non-proteinaceous components using a protease from a bacillus strain.
Invention is credited to Meier, Thomas, Russman, Eberhard, Schmuck, Ranier, Staepels, Johnny, Wehnes, Uwe.
Application Number | 20030165855 10/007389 |
Document ID | / |
Family ID | 26071562 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165855 |
Kind Code |
A1 |
Russman, Eberhard ; et
al. |
September 4, 2003 |
Methods for the analysis of non-proteinaceous components using a
protease from a bacillus strain
Abstract
This invention relates to a method for the analysis of a (at
least one) target non-proteinaceous component of a mixture of
non-proteinaceous and proteinaceous components derived from a
biological sample using a protease from a Bacillus strain. The
invention further relates to a method for the analysis of a (at
least one) target nucleic acid component of a mixture of
non-proteinaceous components, which comprise nucleic acids, and
proteinaceous components whereby the mixture is derived from a
biological sample comprising the steps of incubating the mixture
with a (at least one) protease from a Bacillus strain, optionally
amplifying the (at least one) target nucleic acid component, and
determining or detecting the (at least one) target nucleic acid
component.
Inventors: |
Russman, Eberhard;
(Penzberg, DE) ; Meier, Thomas; (Muenchen, DE)
; Schmuck, Ranier; (Benediktbeueren, DE) ;
Staepels, Johnny; (Weilheim, DE) ; Wehnes, Uwe;
(Ilvesheim, DE) |
Correspondence
Address: |
Pennie & Edmonds, LLP
3300 Hillview Avenue
Palo Alto
CA
94304
US
|
Family ID: |
26071562 |
Appl. No.: |
10/007389 |
Filed: |
October 29, 2001 |
Current U.S.
Class: |
435/6.13 ;
435/68.1 |
Current CPC
Class: |
C12N 9/54 20130101; C12N
15/1003 20130101; G01N 2333/32 20130101; C12Q 1/37 20130101; C12Q
1/6806 20130101; C12Q 2521/537 20130101; C12Q 1/6806 20130101; C12Y
304/21062 20130101 |
Class at
Publication: |
435/6 ;
435/68.1 |
International
Class: |
C12Q 001/68; C12P
021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 2000 |
EP |
EP 00123728-8 |
Mar 15, 2001 |
EP |
EP 01106308-8 |
Claims
1. A method for the analysis of a target non-proteinaceous
component of a mixture of non-proteinaceous and proteinaceous
components derived from a biological sample comprising the step of
incubating the mixture with a protease having an amino acid
sequence which is at least 80% identical to the amino acid sequence
of the protease subtilisin 147 from Bacillus lentus.
2. The method according to claim 1 wherein the amino acid sequence
of the protease is identical to the amino acid sequence of the
protease subtilisin 147 from Bacillus lentus.
3. Themethod according to claim 1 wherein the amino acid sequence
of protease is the amino acid sequence SEQ ID NO 1, a proteolytical
derivative thereof having protease activity or the amino acid
sequence SEQ ID NO 2.
4. The method according to claim 1 wherein the amino acid sequence
of the protease is encoded by the nucleic acid sequence SEQ ID NO
3, a part thereof or a degenerateversion of the nucleic acid
sequence SEQ ID NO 3.
5. The method according to claim 1 wherein the biological sample is
a fluid from the human or animal body.
6. The method according to claim 1 wherein the biological sample is
blood, blood plasma, blood serum or urine.
7. The method according to claim 1 wherein the biological sample
comprises bacterial cells, eukaryotic cells, viruses or mixtures
thereof.
8. The method according to claim 1, wherein after the incubation
step the target non-proteinaceous component is bound to a material
with an affinity thereto, optionally washed and optionally released
from the material with an affinity thereto.
9. The method according to claim 1 wherein the non-proteinaceous
component comprises a nucleic acid.
10. The method according to claim 9 wherein the nucleic acid
comprises DNA or RNA or both.
11. A method for the analysis of a target nucleic acid component of
a mixture comprising the target nucleic acid component, and a
proteinaceous component whereby the mixture is derived from a
biological sample, which method comprising the steps of a)
incubating the mixture with a protease having an amino acid
sequence which is at least 80% identical to the amino acid sequence
of the protease subtilisin 147 from Bacillus lentus, b) optionally
amplifying the target nucleic acid component, and c) determining or
detecting the target nucleic acid component.
12. The method according to claim 11 wherein the amino acid
sequence of the protease is identical to the amino acid sequence of
the protease subtilisin 147 from Bacillus lentus.
13. The method according to claim 11 wherein the amino acid
sequence of protease is the amino acid sequence SEQ ID NO 1, a
proteolytical derivative thereof having protease activity or the
amino acid sequence SEQ ID NO 2.
14. The method according to claim 11 whereinthe amino acid sequence
of the protease is encoded by the nucleic acid sequence SEQ ID NO
3, a part thereof or a degenerated version of the nucleic acid
sequence SEQ ID NO 3.
15. The method according to claim 11 wherein the biological sample
is a fluid from the human or animal body.
16. The method according to claim 11 wherein the biological sample
is blood, blood plasma, blood serum or urine.
17. The method according to claim 11 wherein the target nucleic
acid component comprises DNA or RNA or both.
18. The method according to claim 17 whereinthe DNA or RNA or both
is derived from a virus or a microorganism.
19. The method according to claim 18 wherein the virus is hepatitis
B virus, hepatitis C virus or the human immunodeficiency virus.
20. The method according to claim 11 wherein the target nucleic
acid component is amplified with the polymerase chain reaction.
21. The method according to claim 11 wherein after step a) the
target nucleic acid component is bound to a material with an
affinity to nucleic acids, optionally washed and optionally
released from the material.
22. The method according to claim 21 wherein the material with an
affinity to nucleic acids comprises a material with a silica
surface.
23. The method according to claim 22 whereinthe material with a
silica surface is a glass.
24. The method according to claim 21 whereinthe material with an
affinity to nucleic acids is a composition comprising magnetic
glass particles.
25. The method according to any of the claims 1 to 24, wherein the
analysis is a diagnosis of a disease or a pathogen.
26. A kit comprising a protease having an amino acid sequence,
which is at least 80% identical to the amino acid sequence of the
protease subtilisin 147 from Bacillus lentus.
27. The kit according to claim 26 wherein the amino acid sequence
of the protease is identical to the amino acid sequence of the
protease subtilisin 147 from Bacillus lentus.
28. The kit according to claim 26 wherein the amino acid sequence
of protease is the amino acid sequence SEQ ID NO 1, a proteolytical
derivative thereof having protease activity or the amino acid
sequence SEQ ID NO 2.
29. The kit according to claim 26 wherein the amino acid sequence
of the protease subtilisin 147 is encoded by the nucleic acid
sequence SEQ ID NO 3, a part thereof or a degenerated version of
the nucleic acid sequence SEQ ID NO 3.
30. The kit according to claim 26 wherein it additionally contains
a material with an affinity to nucleic acids.
31. The kit according to claim 30 wherein the material with an
affinity to nucleic acids comprises a material with a silica
surface.
32. The kit according to claim 31 wherein the material with a
silica surface is a glass.
33. The kit according to claim 26 wherein the material with an
affinity to nucleic acids is a composition comprising magnetic
glass particles.
34. The kit according to claim 32 wherein the kit additionally
comprises a lysis buffer, a washing buffer and an elution
buffer.
35. A composition comprising a protease which is at least 80%
identical to the amino acid sequence of the protease subtilisin 147
from Bacillus lentus, in a solution 10 mM Tris acetate pH 5.5, 5 mM
calcium chloride, 5 mM calcium acetate, 1 mM EDTA, and 50% (V/V)
Glycerin.
36. The composition according to claim 35 wherein the amino acid
sequence of the protease is identical to the amino acid sequence of
the protease subtilisin 147 from Bacillus lentus.
37. The composition according to claim 35 wherein the amino acid
sequence of protease is the amino acid sequence SEQ ID NO 1, a
proteolytical derivative thereof having protease activity or the
amino acid sequence SEQ ID NO 2.
38. The composition according to claim 35 wherein the amino acid
sequence of the protease subtilisin 147 is encoded by the nucleic
acid sequence SEQ ID NO 3, a part thereof or a degenerated version
of the nucleic acid sequence SEQ ID NO 3.
Description
[0001] This invention relates to a method for the analysis of a (at
least one) target non-proteinaceous component of a mixture of
non-proteinaceous and proteinaceous components derived from a
biological sample using a protease from a Bacillus strain. The
invention further relates to a method for the analysis of a (at
least one) target nucleic acid component of a mixture of
non-proteinaceous components, which comprise nucleic acids, and
proteinaceous components whereby the mixture is derived from a
biological sample comprising the steps of incubating the mixture
with a (at least one) protease from a Bacillus strain, optionally
amplifying the (at least one) target nucleic acid component, and
determining or detecting the (at least one) target nucleic acid
component.
BACKGROUND ART
[0002] Many biological substances, especially nucleic acids,
present special challenges in terms of isolating them from their
natural environment. On the one hand, they are often present in
very small concentrations and, on the other hand, they are often
found in the presence of many other solid and dissolved substances
e.g. after lysis of cells. This makes them difficult to isolate or
to measure, in particular in biospecific assays which allow the
detection of specific analytes, e.g. nucleic acids, or specific
analyte properties and play a major role in the field of
diagnostics and bioanalytics in research and development. Examples
for biospecific assays are hybridisation assays, immuno assays and
receptor-ligand assays. Hybridisation assays use the specific
base-pairing for the molecular detection of nucleic acid analytes
e.g. RNA and DNA. Hence, oligonucleotide probes with a length of 18
to 20 nucleotides may enable the specific recognition of a selected
complementary sequence e.g. in the human genome. Another assay
which entails the selective binding of two oligonucleotide primers
is the polymerase chain reaction (PCR) described in U.S. Pat. No.
4,683,195. This method allows the selective amplification of a
specific nucleic acid region to detectable levels by a thermostable
polymerase in the presence of desoxynucleotide triphosphates in
several cycles.
[0003] As described above, before the biological substances may be
analysed in one of the above-mentioned assays or used for other
processes, it has to be isolated or purified from biological
samples containing complex mixtures of different components as e.g.
proteinaceous and non-proteinaceous components. Often, for the
first steps, processes are used which allow the enrichment of the
component of interest, e.g. the non-proteinaceous material such as
nucleic acids. Frequently, these are contained in a bacterial cell,
a fungal cell, a viral particle, or the cell of a more complex
organism, such as a human blood cell or a plant cell. The component
of interest can also be called a "target component".
[0004] To release the contents of said cells or particles, they may
be treated with enzymes or with chemicals to dissolve, degrade or
denature the cellular walls of such organisms. This process is
commonly referred to as lysis. The resulting solution containing
such lysed material is referred to as lysate. A problem often
encountered during the lysis is that other enzymes degrading the
non-proteinaceous component of interest, e.g. desoxyribonucleases
or ribonucleases degrading nucleic acids, come into contact with
the component of interest during lysis. These degrading enzymes may
also be present outside the cells or may have been spatially
separated in different cellular compartiments before the lysis and
come now into contact with the component of interest. Other
components released during this process may be e.g. endotoxins
belonging to the family of lipopolysaccharides which are toxic to
cells and can cause problems for products intended to be used in
human or animal therapy.
[0005] There are a variety of means to tackle this problem
mentioned-above. It is common to use chaotropic agents as e.g.
guanidinium thiocyanate or anionic, cationic, zwitterionic or
non-ionic detergents when nucleic acids are intended to be set
free. It is also an advantage to use proteases which rapidly
degrade these enzymes or unwanted proteins. However, this may
produce another problem as the said substances or enzymes can
interfere with reagents or components in subsequent steps.
[0006] Enzymes which can be advantageously used in such lysis or
sample preparation processes mentioned-above are enzymes which
cleave the amide linkages in protein substrates and which are
classified as proteases, or (interchangeably) peptidases (See
Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and
Company, San Francisco, Chapter 3). Proteases which have been used
in the prior art are e.g. alkaline proteases (WO98/04730) or acid
proteases (U.S. Pat. No. 5,386,024). The protease which is widely
used in the prior art for sample preparation for the isolation of
nucleic acids is proteinase K from Tritirachium album (see e.g.
Sambrook et al., 1989) which is active around neutral pH and
belongs to a family of proteases known to the person skilled in the
art as subtilisins. A subtilisin is a serine protease produced by
Gram-positive bacteria or fungi.
[0007] Bacteria of the Bacillus species secrete two extracellular
species of protease, a neutral or metalloprotease, and an alkaline
protease which is functionally a serine endopeptidase, referred to
as subtilisin. A serine protease is an enzyme which catalyzes the
hydrolysis of peptide bonds, in which there is an essential serine
residue at the active site (White, Handler, and Smith, 1973
"Principles of Biochemistry," Fifth Edition, McGraw-Hill Book
Company, N.Y., pp. 271-272). The serine proteases have molecular
weights in the 25,000 to 30,000 Da (Dalton) range. They hydrolyze
simple terminal esters and are similar in activity to eukaryotic
chymotrypsin, also a serine protease. The alternative term,
alkaline protease, reflects the high pH optimum of the serine
proteases, from pH 9.0 to 11.0 (for review, see Priest, 1977,
Bacteriological Rev. 41: 711-753).
[0008] A wide variety of subtilisins have been identified (see e.g.
Kurihara et al., 1972, J. Biol. Chem. 247: 5629-5631; Stahl and
Ferrari, 1984, J. Bacteriol. 158: 411-418; Vasantha et al., 1984,
J. Bacteriol. 159: 811-819, Jacobs et al., 1985, Nucl. Acids Res.
13: 8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366:
421-430; Svendsen et al., 1986, FEBS Lett 196: 228-232; Meloun et
al., 1985, FEBS. Lett. 183: 195-200) including proteinase K from
Tritirachium album (Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler
366: 584-492). Subtilisins are well characterized by their primary
as well as by their tertiary structure (see e.g. Kraut, 1977, Ann.
Rev. Biochem. 46: 331-358; Kurihara et al., 1972, J. Biol. Chem.
247: 5629-5631; Stahl and Ferrari, 1984, J. Bacteriol. 158:
411-418; Vasantha et al., 1984, J. Bacteriol. 159: 811-819; Jacobs
et al., 1985, Nucl. Acids Res. 13: 8913-8926; Nedkov et al., 1985,
Biol. Chem. Hoppe-Seyler 366: 421-430; Svendsen et al., 1986, FEBS
Lett. 196: 228-232; Meloun et al., 1985, FEBS Lett. 183: 195-200;
Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366: 485-492).
[0009] In connection with this invention the amino acid and DNA
sequences of two further serine proteases are of particular
interest. These proteases were derived from two Bacillus lentus
variants, 147 and 309, which have been deposited with NCIB and
designated the accession Nos. NCIB 10147 and NCIB 10309,
respectively (see WO89/06279 and U.S. Pat. No. 3,723,250). For
convenience the proteases produced by these strains are designated
subtilisin 147 and subtilisin 309, respectively, and the genes
encoding these proteins are referred to as the subtilisin 147 and
309 genes. The disclosure of these sequences can be found in
WO89/06279. The equivalents thereto are EP396608 and U.S. Pat. No.
5,741,694. Subtilisins have found much utility in industry,
particularly detergent formulations used for the washing of
clothes.
[0010] In the next steps of the sample preparation which follow on
the lysis step, the component of interest is further enriched. If
the non-proteinaceous components of interest are e.g. nucleic
acids, they are normally extracted from the complex lysis mixtures
before they are used in a probe-based assay.
[0011] There are several methods for the extraction of nucleic
acids:
[0012] sequence-dependent or biospecific methods as e.g.:
[0013] affinity chromatography
[0014] hybridisation to immobilised probes
[0015] sequence-independent or physico-chemical methods as
e.g.:
[0016] liquid-liquid extraction with e.g. phenol-chloroform
[0017] precipitation with e.g. pure ethanol
[0018] extraction with filter paper
[0019] extraction with micelle-forming agents as
cetyl-trimethyl-ammonium-- bromide
[0020] binding to immobilised, intercalating dyes, e.g. acridine
derivatives
[0021] adsorption to silica gel or diatomic earths
[0022] adsorption to magnetic glass particles (MGP) or organo
silane particles under chaotropic conditions
[0023] Particularly interesting for extraction purposes is the
adsorption of nucleic acids to a glass surface although other
surfaces are possible. Many procedures for isolating nucleic acids
from their natural environment have been proposed in recent years
by the use of their binding behavior to glass surfaces.
[0024] As mentioned above, the protease which is widely used in the
prior art for sample preparation for the isolation of nucleic acids
is proteinase K from Tritirachium album. However, this protease has
the disadvantage that the production is relatively expensive.
Further, proteinase K is disadvantageous in methods using magnetic
glass particles for the nucleic acid isolation from EDTA, heparin
or citrate blood plasma, as the particles will often stick to one
another. This is very disadvantageous for automated processes used
for the analysis of a very large number of samples.
[0025] Therefore, it was an object of the present invention to
provide a new method for the analysis of target non-proteinaceous
components, in particular nucleic acids, using a protease which is
relatively cheap, has constant quality and can be used in a variety
of processes. Preferably it should be possible to use it for the
analyis of a (at least one) target nucleic acid component from a
variety of different matrices e.g. EDTA, citrate, or heparin blood
plasma or blood serum. This method should be particularly suitable
in automated processes. Ideally the protease would be also very
active in the presence of chaotropic agents frequently used in the
processes for the purification of nucleic acids.
[0026] This problem was solved by the findings of the present
invention which is related to a method for the analysis of a (at
least one) target non-proteinaceous component of a mixture of
non-proteinaceous and proteinaceous components derived from a
biological sample comprising the step of incubating the mixture
with a (at least one) protease having an amino acid sequence which
is at least 80% identical to the amino acid sequence of the
protease subtilisin 147 from Bacillus lentus. As can be seen from
the example, the protease is very active in the presence of
chaotropic agents or equally active for the digestion of citrate or
EDTA blood plasma. This could not be foreseen from the prior
art.
[0027] In summary, this invention relates to a method for the
analysis of a (at least one) target non-proteinaceous component of
a mixture of non-proteinaceous and proteinaceous components derived
from a biological sample using a protease from a Bacillus strain.
The invention further relates to a method for the analysis of a (at
least one) target nucleic acid component of a mixture of
non-proteinaceous components, which comprise nucleic acids, and
proteinaceous components whereby the mixture is derived from a
biological sample comprising the steps of incubating the mixture
with a (at least one) protease having an amino acid sequence which
is at least 80% identical to the amino acid sequence of the
protease subtilisin 147 from Bacillus lentus, optionally amplifying
the (at least one) target nucleic acid component, and determining
or detecting the (at least one) target nucleic acid component.
Optionally, the nucleic acids and the (at least one) target nucleic
acid component are bound to a material with an affinity thereto,
optionally washed and optionally released from the material with an
affinity thereto, whereby the material with an affinity to nucleic
acids and the (at least one) target nucleic acid component
comprises a material with a silica surface, in particular magnetic
glass particles. The invention is further related to the use of a
protease according to the invention in diagnostics, research and
bioanalytics e.g. for the purification of nucleic acids, for the
analysis of a (at least one) target non-proteinaceous component of
a mixture of non-proteinaceous and proteinaceous components derived
from a biological sample, for the enrichment of a (at least one)
target non-proteinaceous component of a mixture of
non-proteinaceous and proteinaceous components derived from a
biological sample or for the purification or isolation of a (at
least one) target non-proteinaceous component of a mixture of
non-proteinaceous and proteinaceous components derived from a
biological sample. The invention is also related to a kit
comprising the protease according to the invention and the use of a
kit according to the invention in diagnostics and/or for the
purification of nucleic acids. The invention will be described in
more detail below.
[0028] Figure legends:
[0029] FIG. 1a: Comparison of the digestion of EDTA plasma versus
citrate plasma with Esperase as analyzed by high pressure liquid
chromatography
[0030] FIG. 1b: Comparison of the digestion of EDTA plasma versus
citrate plasma with proteinase K as analyzed by high pressure
liquid chromatography
[0031] FIG. 2: Determination of the pH Optimum of Esperase
[0032] FIG. 3: Determination of the residual activity of Esperase
versus proteinase K in dependence of the concentration of a
chaotropic agent. The highest activity is set to a value of 100%
and the other concentrations are calculated relative to the highest
value.
[0033] FIG. 4: Determination of the stability of Esperase versus
proteinase K in dependence from the concentration of guanidinium
thiocyanate. The activity of the protease is measured directly
after the addition of guanidinium thiocyanate and after 15 min at
25.degree. C. in the presence of guanidinium thiocyanate. The
percentage of the residual activity at different guanidinium
thiocyanate concentrations is shown in this figure.
[0034] FIG. 5: Stability in Storage Buffer (composition: 10 mM Tris
acetate, 5 mM calcium chloride, 5 mM calcium acetate, 1 mM EDTA,
50% (V/V) Glycerin with a pH value of 5.5) of esperase versus
proteinase K
DESCRIPTION OF THE INVENTION
[0035] It is one embodiment of this invention to provide a method
for the analysis of a (at least one) target non-proteinaceous
component of a mixture of non-proteinaceous and proteinaceous
components derived from a biological sample comprising the step of
incubating the mixture with a (at least one) protease having an
amino acid sequence which is at least 80% identical to the amino
acid sequence of the protease subtilisin 147 from Bacillus lentus.
The term "derived" means that a biological sample is manipulated or
treated in order to create a mixture of non-proteinaceous and
proteinaceous components which are originally contained in the
biological sample. From this mixture it should be possible to
analyse, isolate, enrich or purify specific non-proteinaceous
components. The term "analysis" shall mean that the presence or the
amount of the target non-proteinaceous component is investigated,
i.e. the target non-proteinaceous component is detected or
determined or the amount thereof is determined. Manipulation or
treatment steps include chemical or physical manipulation steps
which are known to the expert in the field. More specifically, this
can be done by lysing the biological sample. Biological samples are
samples which are taken from a plant or an animal (including a
human being) and are solid or liquid. Specific examples are
described in more detail below.
[0036] In a further embodiment of the invention, the method has
further steps after the incubation as binding the (at least one)
target non-proteinaceous component to a material with an affinity
thereto, optionally washing and optionally releasing the (at least
one) target non-proteinaceous component from the material with an
affinity thereto. Afterwards, the (at least one) target
non-proteinaceous component may be determined or detected by
standard analytical methods known to the person skilled in the art
and described e.g. in Sambrook et al. (1989), Molecular Cloning,
Cold Spring Harbor University Press, New York, N.Y., USA or in
"Bioanalytik", Lottspeich and Zorbas (eds.), 1.sup.st edition 1998,
Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany.
Preferably, the amount of the target non-proteinaceous component is
determined with the methods described therein. The method according
to the invention is preferably used in research, bioanalytics in
particular in diagnostics or in diagnostic investigations in
medicine, i.e. in methods that are used to determine the cause of
an illness or disorder in humans or in animals.
[0037] Therefore, a preferred embodiment of the invention is a
method for the analysis of a (at least one) target
non-proteinaceous component of a mixture of non-proteinaceous and
proteinaceous components derived from a biological sample
comprising the steps of:
[0038] a) incubating the mixture with a (at least one) protease
according to the invention,
[0039] b) binding the (at least one) target non-proteinaceous
component to a material with an affinity thereto,
[0040] c) optionally washing and optionally releasing the (at least
one) target non-proteinaceous component from the material with an
affinity thereto, and
[0041] d) determining or detecting the (at least one) target
non-proteinaceous component.
[0042] In the most preferred embodiment, the step c) is not
optional, i.e. that the bound (at least one) target
non-proteinaceous component is washed and released from the
material with an affinity thereto. Preferably the amount of the
target non-proteinaceous component is determined.
[0043] The protease according to the invention degrades the
proteinaceous components, i.e. the components containing peptide
bonds which shall be hydrolyzed if it is of interest to enrich,
isolate or purify the (at least one) target non-proteinaceous
component of the biological sample. The protease according to the
present invention may be added in solid form e.g. as a tablet or a
powder or in a dissolved form in a buffered or unbuffered solution
in a similar manner as described for proteinase K.
[0044] For the purpose of this invention, the term "esperase" shall
mean the protease according to the invention, i.e. the protease
subtilisin 147 derived from the Bacillus lentus variant 147, which
was deposited with NCEB under accession No. NCIB 10147. The amino
acid sequence SEQ ID NO 1 is the full length amino acid sequence of
the protease subtilisin 147 (or esperase) including a signal
sequence which is removed after secretion by the action of
proteases. A signal sequence is a sequence that directs secretion
of an expressed protein from the host cell and is proteolytically
removed after secretion. The SEQ ID NO 2 is the sequence of
esperase without signal sequence. The term "esperase" shall also
comprise those proteolytical derivatives of SEQ ID NO 1 which might
be generated by incomplete or inexact processing of the signal
sequence and which still have proteolytic activity including those
with a lower activity than that of the correctly processed
esperase. The amino acid sequence of the protein may be encoded by
the subtilisin 147 gene, i.e. the nucleotide sequence SEQ ID NO 3,
by parts thereof or a degenerated version thereof. Degenerated
sequences are degenerated within the meaning of the genetic code in
that an unlimited number of nucleotides are replaced by other
nucleotides without resulting in a change of the amino acid
sequence originally encoded.
[0045] According to the present invention the term "proteinaceous
material" is meant to describe material that contains a (at least
one) peptide bond, therefore "proteinaceous material" is preferably
a composition of matter containing a (at least one) protein with
natural amino acids. Most of these peptide bonds may be hydrolyzed
by the protease according to the present invention depending on the
chemical nature of the neighboring chemical groups (or amino acids)
and the accessibility of the peptide bond, i.e. the proteinaceous
material is a substrate to the protease according to the invention.
In consequence, the term "non-proteinaceous material" is meant to
describe material that does not contain a peptide bond and is not
substrate to the protease according to the present invention.
[0046] The protease subtilisin 147 from Bacillus lentus is
commercially available e.g. from Roche Molecular Biochemicals,
Mannheim, Germany, or from Novo Nordisk, Denmark. In certain
embodiments of the invention, the mixture of proteinaceous
components and target non-proteinaceous components can be incubated
with commercially available protease without any purification or
preparation of the protease. In other embodiments of the invention,
the commercially available protease can be prepared or purified to
remove contaminants. For example, the protease can be dialyzed
against a buffer that is compatible with the mixture. A solution
comprising the commercially availiable protease can also be
filtered or sterilized. In addition, the commercially available
protease can optionally be purified or partially purified to remove
contaminants such as nucleases according to protein purification
techniques known to those of skill in the art. For instance, the
commercially available protease can be partially purified by
ammonium sulfate precipitation and/or by chromatography methods
such as heparin-sepharose ff chromatography. Preferred methods for
the preparation of commercially available subtilisin 147 are
described in the examples below.
[0047] Another possibility to obtain this protease is to isolate
the gene from the deposited microorganism or to synthesize the gene
coding for that protease according to standard methodology see e.g.
Sambrook et al. (1989), Molecular Cloning, Cold Spring Harbor
University Press, New York, N.Y., USA. The amino acid sequence of
the pro-protein comprising a signal sequence (SEQ ID NO 1), the
amino acid sequence of the secreted protease (SEQ ID NO 2) and the
DNA sequence (see SEQ ID NO 3) of this protein are known from
WO89/06279, EP 396 608 and WO98/20115. The major form of the
secreted protein is encoded by the nucleotides 280 to 1083 of SEQ
ID NO 3, i.e. the signal peptide is encoded by the nucleotides 1 to
279 of SEQ ID NO 3. The isolation of the microorganism is described
in U.S. Pat. No. 3,723,250. The isolated strain is deposited under
NCIB 10147. Custom gene synthesis can be performed by example by
Operon Technologies, Alameda, Calif., USA, recently acquired by
Qiagen, Germany. Using standard methodology the person skilled in
the art can construct an expression vector, express the gene
product and isolate the protein essentially as described in
WO89/06279 or WO98/20115 which shall be incorporated herein by
reference.
[0048] With this information in hand, the expert in the field can
also construct and express a gene coding for a protease with an
amino acid sequence with 80% identity to the amino acid sequence of
subtilisin 147 by substituting various amino acids. Therefor, he
uses standard methodology as described in Sambrook et al. (1989),
Molecular Cloning, Cold Spring Harbor University Press, New York,
N.Y., USA or methodology as described in WO89/06279 or WO98/20115.
The tests for the proteolytical activity are described in these two
international applications or in this invention.
[0049] In further embodiments, a method according to the invention
is disclosed in which a protease is used with an amino acid
sequence which is identical (100% identical) to the amino acid
sequence of the protease subtilisin 147 from Bacillus lentus. In a
further embodiment, a method according to the invention is
disclosed characterized in that the amino acid sequence of protease
is the amino acid sequence SEQ ID NO 1, a proteolytical derivative
thereof having protease activity or the amino acid sequence SEQ ID
NO 2. In still another embodiment of the invention, a method
according to the invention is disclosed characterized in that the
amino acid sequence of the protease according to the invention is
encoded by the nucleic acid sequence SEQ ID NO 3, a part thereof
coding for an active protease according to the invention or a
degenerated version of the nucleic acid sequence SEQ ID NO 3. The
invention contemplates derivatives of the DNA sequence SEQ ID NO 3
which have been altered by substitutions, deletions and additions
that provide for functionally equivalent molecules. For example,
due to the degeneracy of nucleotide coding sequences, other DNA
sequences which encode substantially the same amino acid sequence
as depicted in SEQ ID NO 1 or 2 can be used in the practice of this
invention. Further, amino acid sequences can be used which have
amino acid substitutions at positions where amino acids of the same
group, e.g. polar or hydrophobic have been exchanged for one
another.
[0050] In an embodiment of the invention the biological sample is
intended to comprise viruses or bacterial cells, as well as
isolated cells from multicellular organisms as e.g. human and
animal cells such as leucocytes, and immunologically active low and
high molecular chemical compounds such as haptens, antigens,
antibodies and nucleic acids, blood plasma, cerebral fluid, sputum,
stool, biopsy specimens, bone marrow, oral rinses, blood serum,
tissues, urine or mixtures thereof. In a preferred embodiment of
the invention the biological sample is a fluid from the human or
animal body, preferably the biological sample is blood, blood
plasma, blood serum or urine. The blood plasma is preferably EDTA,
heparin or citrate blood plasma. In an embodiment of the invention
the biological sample comprises bacterial cells, eukaryotic cells,
viruses or mixtures thereof.
[0051] The biological sample can also be of a type used for
environmental analysis, food analysis or molecular biology
research, e.g. from bacterial cultures, phage lysates. In certain
cases the sample can be used without pretreatment in the method
according to the invention. In many cases, however, the sample
should be lysed using an appropriate method, releasing the
biological substances contained in the sample thereby creating a
mixture of proteinaceous and non-proteinaceous components derived
from the biological sample. Procedures for lysing samples are known
by the expert and can be chemical, enzymatic or physical in nature.
A combination of these procedures is applicable as well. For
instance, lysis can be performed using ultrasound, high pressure,
by shear forces, using alkali, detergents or chaotropic saline
solutions, or by means of proteases or lipases. With regard for the
lysis procedure to obtain nucleic acids, special reference is made
to Sambrook et al.: Molecular Cloning, A Laboratory Manual, 2nd
Addition, Cold Spring Harbour Laboratory Press, Cold Spring
Harbour, NY, USA, and Ausubel et al.: Current Protocols in
Molecular Biology 1987, J. Wiley and Sons, NY, USA.
[0052] In still another embodiment of the invention the biological
sample comprises a (at least one) glycosylated protein which is
partially or fully degraded by the protease according to the
invention. Therefore, the invention also contemplates the use of
the protease according to the invention for the partial or full
degradation of glycosylated proteins, i.e. proteins with covalently
attached carbohydrate moieties.
[0053] The method according to the invention can also have further
steps after the incubation as binding the (at least one) target
non-proteinaceous component to a material with an affinity thereto,
optionally washing and optionally releasing the (at least one)
target non-proteinaceous component from the material with an
affinity thereto. Afterwards, the (at least one) target
non-proteinaceous component may be determined or detected by
standard analytical methods known to the person skilled in the art
and described e.g. in Sambrook et al. (1989), Molecular Cloning,
Cold Spring Harbor University Press, New York, N.Y., USA or in
"Bioanalytik", Lottspeich and Zorbas (eds.), 1.sup.st edition 1998,
Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany.
[0054] In order to bind the (at least one) target non-proteinaceous
component to a material with an affinity thereto, the mixture of
non-proteinaceous and proteinaceous components is brought in
contact with the material with an affinity to the (at least one)
target non-proteinaceous component under conditions in which the
(at least one) target non-proteinaceous component binds to the
surface of the material. The conditions for this depend on the type
of the (at least one) target non-proteinaceous component involved,
but are basically known to the expert in the field. They also
depend on the method by which the (at least one) target
non-proteinaceous component is bound to the surface. For example,
if modified nucleic acids are the target non-proteinaceous
components, the binding can take place via the groups of nucleic
acids that represent the modification, e.g., biotin via binding
with streptavidin-coated surfaces.
[0055] If unmodified nucleic acids are the target non-proteinaceous
components, a direct binding of the nucleic acids to a material
with a silica surface is preferred because among other reasons the
nucleic acids do not have to be modified and even native nucleic
acids can be bound. These processes are described in detail by
various documents. In Proc. Natl. Acad. USA 76, 615-691 (1979), for
instance, a procedure for binding nucleic acids from agarose gels
in the presence of sodium iodide to ground flint glass is proposed.
The purification of plasmid DNA from bacteria on glass dust in the
presence of sodium perchlorate is described in Anal. Biochem. 121,
382-387 (1982). In DE-A 37 34 442, the isolation of single-stranded
M13 phage DNA on glass fiber filters by precipitating phage
particles using acetic acid and lysis of the phage particles with
perchlorate is described. The nucleic acids bound to the glass
fiber filters are washed and then eluted with a methanol-containing
Tris/EDTA buffer. A similar procedure for purifying DNA from lambda
phages is described in Anal. Biochem. 175, 196-201 (1988). The
procedure entails the selective binding of nucleic acids to glass
surfaces in chaotropic salt solutions and separating the nucleic
acids from contaminants such as agarose, proteins or cell residue.
To separate the glass particles from the contaminants, the
particles may be either centrifuged or fluids are drawn through
glass fiber filters. This is a limiting step, however, that
prevents the procedure from being used to process large quantities
of samples. The use of magnetic particles to immobilize nucleic
acids after precipitation by adding salt and ethanol is more
advantageous and described e.g. in Anal. Biochem. 201, 166-169
(1992) and PCT GB 91/00212. In this procedure, the nucleic acids
are agglutinated along with the magnetic particles. The agglutinate
is separated from the original solvent by applying a magnetic field
and performing a wash step. After one wash step, the nucleic acids
are dissolved in a Tris buffer. This procedure has a disadvantage,
however, in that the precipitation is not selective for nucleic
acids. Rather, a variety of solid and dissolved substances are
agglutinated as well. As a result, this procedure can not be used
to remove significant quantities of any inhibitors of specific
enzymatic reactions that may be present. Magnetic, porous glass is
also available on the market that contains magnetic particles in a
porous, particular glass matrix and is covered with a layer
containing streptavidin. This product can be used to isolate
biological materials, e.g., proteins or nucleic acids, if they are
modified in a complex preparation step so that they bind covalently
to biotin. Magnetizable particular adsorbents proved to be very
efficient and suitable for automatic sample preparation.
Ferrimagnetic and ferromagnetic as well as superparamagnetic
pigments are used for this purpose. The most preferred MGPs are
those described in WO01/37291.
[0056] In detail, the procedure for binding the (at least one)
target nucleic acid to glass particles can be described as follows.
It is preferably performed in the presence of chaotropic salts with
a concentration of between 1 and 8 mol/l, and preferably between 2
and 6 mol/l. Chaotropic salts can be sodium iodide, sodium
perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate or
guanidinium hydrochloride. Other substances are also possible. The
purification effect results from the behavior of DNA or RNA to bind
to material with a glass surface under these conditions i.e. in the
presence of certain concentration of a chaotropic agent, higher
concentrations of organic solvents or under acidic conditions. To
bring the sample in contact with the material with an affinity to
the (at least one) target non-proteinaceous component, the sample
is mixed with the material and incubated for a period of time
sufficient for the binding to occur. Experts are usually familiar
with the duration of the incubation step from procedures for
performing treatment with non-magnetic particles. This step can be
optimized by determining the quantity of immobilized biological
material on the surface at different points in time. Incubation
times of between 10 seconds and 30 minutes can be appropriate for
nucleic acids. After incubation, the bound (at least one) target
non-proteinaceous component is separated from the liquid. This may
be achieved in general by gravity or in the convenient case of
nucleic acids bound to magnetic glass particles by separating the
material bound to the magnetic particles by applying a magnetic
field. For instance, the magnetic particles can be pulled to the
wall of the vessel in which incubation was performed. The liquid
containing the sample contents that were not bound to the magnetic
particles can then be removed. The removal procedure used depends
on the type of vessel in which incubation was performed. Suitable
steps include removing the liquid via pipetting or aspiration. The
material with the bound DNA or RNA may then be washed at least
once, preferably with a mixture of 70 volume parts ethanol with 30
volume parts water ("70% Ethanol"). A wash solution is used that
does not cause the (at least one) target non-proteinaceous
component to be released from the material surface but that washes
away the undesired contaminants as thoroughly as possible. This
wash step preferably takes place by incubating the material with
the bound (at least one) target non-proteinaceous component with
the wash solution. The material is preferably resuspended during
this step. The contaminated wash solution is preferably removed
just as in the step described above for binding the biological
material. After the last wash step, the material can be dried
briefly in a vacuum, or the fluid can be allowed to evaporate. A
pretreatment step using acetone may also be performed. Afterwards,
the conditions may be reversed, e.g. the concentration of the
chaotropic agent or organic solvent is decreased to elute the DNA
or RNA bound to the material. Preferably, the process of separating
the magnetic glass particles from the rest of the sample is done by
pelleting the immobilized biological material, e.g. by gravity
force or by the use of a magnet in the case of magnetic glass
particles and removal of the supernatant. Then the magnetic glass
particles with the immobilized biological material are resuspended
in a solution with no or only a low amount of chaotropic agent
and/or organic solvent. Alternatively, the suspension can be
diluted with a solution with no or only a low amount of chaotropic
agent and/or organic solvent. Buffers of this nature are known from
DE 3724442 and Analytical Biochemistry 175, 196-201 (1988). The
elution buffers with a low salt content are in particular buffers
with a content of less than 0.2 mol/l. In an especially preferred
embodiment, the elution buffer contains the substance Tris for
buffering purposes. In another special embodiment, the elution
buffer is demineralized water. The solution containing purified DNA
or RNA can now be used for other reactions.
[0057] For washing and binding steps, preferably liquids are used
which are suitable for processes in molecular biology, in
particular desoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
purification processes which make use of the binding of these
substances to glass particles under certain conditions. Preferred
liquids comprise alcohols and/or ketones or any mixtures thereof
with water. Alcohols shall include according to the invention
preferably primary, secondary or tertiary alcohols of the general
formula R-OH where the R stands for the general formula
--(--CH.sub.2).sub.n--CH.sub.3 with n>=0. However, other
alcohols can also be used if they are suitable for molecular
biology purposes as e.g. glycerol. Particularly suitable are the
alcohols isopropanol, ethanol or mixtures thereof with water,
preferably a mixture of 80 volume parts of isopropanol with 20
volume parts of water. In another embodiment of the invention the
liquid comprises ketones as e.g. acetone.
[0058] The magnetic glass particles used in the present invention
may be provided in different formulations. It is possible to
provide them in the form of a tablet, as a powder or preferably as
a suspension. In a preferred embodiment of the invention these
suspensions contain between 5 to 60 mg/ml magnetic glass particles
(MGPs). In another embodiment of the invention the
silica-containing material is suspended in aqueous buffered
solutions which may optionally contain a chaotropic agent in a
concentration of between 2 and 8 mol/l, and preferably between 4
and 6 mol/l. Chaotropic salts are sodium iodide, sodium
perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate or
guanidinium hydrochloride. Other compounds known to the expert in
the field are also possible. A chaotropic agent according to the
present invention is any chemical substance which disturbs the
ordered structure of liquid water and has the effect that DNA or
RNA binds to the magnetic glass particles if this agent is present
in the DNA or RNA containing solution. It is obvious for the
artisan to produce suitable aqueous buffered solutions. Buffer
systems which suitable for molecular biology purposes may be found
e.g. in Sambrook et al. (1989), Molecular Cloning, Cold Spring
Harbor University Press, New York, N.Y., USA. Preferred buffer
substances are Tris-(hydroxymethyl)-aminomethane (TRIS), phosphate,
N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES),
salts thereof or other suitable substances. Additionally,
substances may be present which modify the ionic strength of the
solution as e.g. NaCl, KCl or CaCl.sub.2 or which are metal cation
complexing agents as e.g. ethylene-diamine-tetra-acetic acid (EDTA)
or the salts thereof. Other biological substances known to the
expert in the field may also be present. The method according to
the present invention is suitable for the purification of nucleic
acids, i.e. RNA or DNA, from complex mixtures with other biological
substances containing them. Thereby also mixtures of different
nucleic acids may be purified, even mixtures containing a nucleic
acid of interest in low abundance. In one embodiment of the
invention mixtures of specific nucleic acids are purified, in which
the target nucleic acid(s) may be a minor component in terms of
concentration (or may be present in low abundance).
[0059] The procedure described can be used to isolate native or
modified biological material. Native biological material is
understood to be material, the structure of which was not
irreversibly changed compared with the naturally-occurring
biological materials. This does not mean that other components of
the sample can not be modified, however. Modified biological
materials include materials that do not occur in nature, e.g.,
nucleic acids that are modified by attaching to them groups that
are reactive, detectable or capable of immobilization. An example
of this are biotinylated nucleic acids.
[0060] After the steps described above, the non-proteinaceous
components isolated using the method according to the invention can
now be used further as necessary. For instance, they can be used as
a substrate for various enzymatic reactions. When nucleic acids are
involved, they can be used for sequencing, radioactive or
non-radioactive labelling, amplification of one or more of the
sequences they contain, transcription, hybridization with labelled
probe nucleic acids, translation or ligation. Therefore, in a more
preferred embodiment of the invention the method comprises the step
of releasing the bound (at least one) target non-proteinaceous
component from the material with an affinity thereto. If desired,
the (at least one) target non-proteinaceous component purified in
this manner can be separated from the material as described
above.
[0061] In a preferred embodiment of the invention the method
comprises the step of detecting or determining a (at least one)
target non-proteinaceous component. A preferred embodiment of the
invention are therefore the above-described purification method
followed by a determination or detection step or purification
methods followed by an amplification and determination or detection
step. In the case of nucleic acids, the target nucleic acid or
nucleic acids of interest may be contained in a matrix of
non-target nucleic acids, and may even be a minor component in said
mixture of specific nucleic acids. Suitable DNA detection methods
are known to the expert in the field and are described in standard
textbooks as Sambrook et al.: Molecular Cloning, A Laboratory
Manual, 2nd Addition, Cold Spring Harbour Laboratory Press, Cold
Spring Harbour, NY and Ausubel et al.: Current Protocols in
Molecular Biology 1987, J. Wiley and Sons, NY. There may be also
further purification steps before the DNA detection step is carried
out as e.g. a precipitation step. The detection methods may include
but are not limited to the binding or intercalating of specific
dyes as ethidiumbromide which intercalates into the double-stranded
DNA and changes its fluorescence thereafter. The purified DNA may
also be separated by electrophoretic methods optionally after a
restriction digest and visualized thereafter. There are also
probe-based assays which exploit the oligonucleotide hybridisation
to specific sequences and subsequent detection of the hybrid. It is
also possible to sequence the DNA after further steps known to the
expert in the field. Other methods apply a diversity of DNA
sequences to a silicon chip to which specific probes are bound and
yield a signal when a complementary sequences bind.
[0062] In a preferred embodiment of the invention the mixture of
non-proteinaceous and proteinaceous components comprises nucleic
acids whereby the nucleic acids comprise DNA or RNA or both.
[0063] A preferred embodiment of the invention is related to a
method for the analysis of a (at least one) target nucleic acid
component of a mixture non-proteinaceous components, which comprise
nucleic acids, and proteinaceous material derived from a biological
sample comprising the steps of
[0064] a) incubating the mixture with a (at least one) protease
having an amino acid sequence which is at least 80% identical to
the amino acid sequence of the protease subtilisin 147 from
Bacillus lentus,
[0065] b) optionally amplifying the (at least one) target nucleic
acid component, and
[0066] c) determining or detecting the (at least one) target
nucleic acid component.
[0067] In a preferred embodiment of the invention, the amount of
the target nucleic acid component is determined.
[0068] In an embodiment of the invention the amino acid sequence of
the protease is identical to the amino acid sequence of the
protease subtilisin 147 from Bacillus lentus. In a preferred
embodiment of the invention the amino acid sequence of protease is
the amino acid sequence SEQ ID NO 1, a proteolytical derivative
thereof having protease activity or the amino acid sequence SEQ ID
NO 2. In yet another preferred embodiment of the invention the
amino acid sequence of the protease according to the invention is
encoded by the nucleic acid sequence SEQ ID NO 3, a part thereof or
a degenerated version of the nucleic acid sequence SEQ ID NO 3. In
still another embodiment of the invention the biological sample is
intended to comprise viruses or bacterial cells, as well as
isolated cells from multicellular organisms as e.g. human and
animal cells such as leucocytes, and immunologically active low and
high molecular chemical compounds such as haptens, antigens,
antibodies and nucleic acids, blood plasma, cerebral fluid, sputum,
stool, biopsy specimens, bone marrow, oral rinses, blood serum,
tissues, urine or mixtures thereof In a preferred embodiment of the
invention the biological sample is a fluid from the human or animal
body, preferably the biological sample is blood, blood plasma,
blood serum or urine. The blood plasma is preferably EDTA, heparin
or citrate blood plasma. In an embodiment of the invention the
biological sample comprises bacterial cells, eukaryotic cells,
viruses or mixtures thereof.
[0069] In a preferred embodiment of the invention the mixture of
nucleic acids and proteinaceous material comprises
desoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or both,
preferably the DNA or RNA or both is derived from a (at least one)
virus or a (at least one) microorganism. The virus can be hepatitis
A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV),
the human immunodeficiency virus (HIV), the human papilloma virus
(HPV) or parvovirus B 19.
[0070] In a preferred embodiment of the invention a (at least one)
target nucleic acid component and the other nucleic acids are
purified essentially as described above. Then the (at least one)
target nucleic acid component is further manipulated and detected,
i.e. it is amplified with the polymerase chain reaction which
specifically amplifies target sequences to detectable amounts.
Other possible amplification reactions are the ligase Chain
Reaction (LCR, Wu and Wallace, 1989, Genomics 4:560-569 and Barany,
1991, Proc. Natl. Acad. Sci. USA 88:189-193); Polymerase Ligase
Chain Reaction (Barany, 1991, PCR Methods and Applic. 1:5-16);
Gap-LCR(PCT Patent Publication No. WO 90/01069); Repair Chain
Reaction (European Patent Publication No. 439,182 A2), 3SR (Kwoh et
al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177; Guatelli et
al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878; PCT Patent
Publication No. WO 92/0880A), and NASBA (U.S. Pat. No. 5,130,238).
Further, there are strand displacement amplification (SDA),
transciption mediated amplification (TMA), and
Q.beta.-amplification (for a review see e.g. Whelen and Persing
(1996). Annu. Rev. Microbiol. 50, 349-373; Abramson and Myers,
1993, Current Opinion in Biotechnology 4:41-47).
[0071] A particularly preferred detection method is the TaqMan.RTM.
method disclosed in WO92/02638 and the corresponding US patents
U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375, U.S. Pat. No.
5,487,972. This method exploits the exonuclease activity of a
polymerase to generate a signal. In detail, the (at least one)
target nucleic acid component is detected by a process comprising
contacting the sample with an oligonucleotide containing a sequence
complementary to a region of the target nucleic acid component and
a labeled oligonucleotide containing a sequence complementary to a
second region of the same target nucleic acid component sequence
strand, but not including the nucleic acid sequence defined by the
first oligonucleotide, to create a mixture of duplexes during
hybridization conditions, wherein the duplexes comprise the target
nucleic acid annealed to the first oligonucleotide and to the
labeled oligonucleotide such that the 3'-end of the first
oligonucleotide is adjacent to the 5'-end of the labeled
oligonucleotide. Then this mixture is treated with a
template-dependent nucleic acid polymerase having a 5' to 3'
nuclease activity under conditions sufficient to permit the 5' to
3' nuclease activity of the polymerase to cleave the annealed,
labeled oligonucleotide and release labeled fragments.
[0072] The signal generated by the hydrolysis of the labeled
oligonucleotide is detected and/or measured. TaqMan.RTM. technology
eliminates the need for a solid phase bound reaction complex to be
formed and made detectable. In more general terms, a procedure for
the purification of a (at least one) target nucleic acid component
followed by a detection step is disclosed wherein the amplification
and/or detection reaction is a homogeneous solution-phase.
[0073] In another preferred embodiment of the invention the nucleic
acids including the (at least one) target nucleic acid component
are bound to a material with an affinity thereto before they are
optionally amplified or determined or detected. After binding they
are optionally washed and optionally released from the material
with an affinity thereto essentially as described above. Therefore,
a preferred embodiment of the invention is related to a method for
the analysis of a (at least one) target nucleic acid component of a
mixture non-proteinaceous components, which comprise nucleic acids,
and proteinaceous material derived from a biological sample
comprising the steps of
[0074] a) incubating the mixture with a (at least one) protease
according to the invention
[0075] b) binding the (at least one) target non-proteinaceous
component to a material with an affinity thereto,
[0076] c) optionally washing and optionally releasing the (at least
one) target nucleic acid component from the material with an
affinity thereto,
[0077] d) optionally amplifying the (at least one) target nucleic
acid component, and
[0078] e) determining or detecting the (at least one) target
nucleic acid component.
[0079] In the most preferred embodiment, the steps c) and d) are
not optional, i.e. that the bound (at least one) target nucleic
acid component is washed and released from the material with an
affinity thereto and the (at least one) target nucleic acid
component is amplified before it is determined or detected.
Preferably the amount of the target nucleic acid component is
determined.
[0080] The material with an affinity to nucleic acids and the (at
least one) target nucleic acid component comprises a material with
a silica surface, preferably the material with a silica surface is
a glass, most preferably the material with an affinity to nucleic
acids is a composition comprising magnetic glass particles. The
steps are performed essentially as already describe above. In
summary, magnetic glass particles are added to the lysis mixture
comprising the nucleic acids including the (at least one) target
nucleic acid component. After a suitable period of time for
adsorption to take place--which can be optimized by mechanical
agitation--the particles are separated from the surrounding fluid
that contains additional components that are not to be detected.
This is performed preferably by applying a magnetic field by
placing a magnet against the vessel wall and removing the remaining
liquid from the tube. To remove further contaminants that may still
be present, a wash step is preferably performed with a fluid that
does not cause the nucleic acids and the (at least one) target
nucleic acid component to be released from the glass surface. An
elution buffer having reagent conditions under which the nucleic
acids and the (at least one) target nucleic acid component are not
bound to the glass surface and are eluted is added to remove the
nucleic acids including the (at least one) target nucleic acid
component from the glass surface. These conditions are low salt
conditions in particular. Depending on the intended further use of
the nucleic acids and the (at least one) target nucleic acid
component, the fluid can now be separated from the particles and
processed further. This separation step is preferably performed via
application of a magnetic field so that the particles are separated
from the eluate. The most preferred magnetic glass particles for
this method are described in WO01/37291.
[0081] Preferably the method according to the invention is used for
diagnostic analysis or bioanalytics.
[0082] In a preferred embodiment of the invention the protease
according to the invention is used in research, bioanalytics or
diagnostics. In further preferred embodiments the protease
according to the invention is used for the analysis of a (at least
one) target non-proteinaceous component of a mixture of
non-proteinaceous and proteinaceous components derived from a
biological sample, for the enrichment of a (at least one) target
non-proteinaceous component of a mixture of non-proteinaceous and
proteinaceous components derived from a biological sample or for
the purification or isolation of a (at least one) target
non-proteinaceous component of a mixture of non-proteinaceous and
proteinaceous components derived from a biological sample.
Preferably the (at least one) target non-proteinaceous component is
a nucleic acid, preferably from a virus or a microorganism, or the
mixture of non-proteinaceous and proteinaceous components comprises
nucleic acids. Preferred viruses are hepatitis B virus, hepatitis C
virus or the human immunodeficiency virus or the other viruses
described above.
[0083] The invention further contemplates a kit of parts
characterized in that it contains a (at least one) protease having
an amino acid sequence, which is at least 80% identical to the
amino acid sequence of the protease subtilisin 147 from Bacillus
lentus. In another embodiment of the invention the amino acid
sequence of the protease is identical to the amino acid sequence of
the protease subtilisin 147 from Bacillus lentus. In a preferred
embodiment of the invention the amino acid sequence of protease is
the amino acid sequence SEQ ID NO 1, a proteolytical derivative
thereof having protease activity or the amino acid sequence SEQ ID
NO 2, preferably the amino acid sequence of the protease according
to the invention is encoded by the nucleic acid sequence SEQ ID NO
3, a part thereof coding for an active protease or a degenerated
version of the nucleic acid sequence SEQ ID NO 3. Such kits known
in the art further comprise plastics ware which can be used during
the sample preparation procedure as e.g. microtitre plates in the
96 or 384 well format or just ordinary reaction tubes manufactured
e.g. by Eppendorf, Hamburg, Germany and all other reagents for
carrying out the method according to the invention. Therefore, the
kit can additionally contain a material with an affinity to nucleic
acids (and the (at least one) target nucleic acid component),
preferably the material with an affinity to nucleic acids (and the
(at least one) target nucleic acid component) comprises a material
with a silica surface. Preferably, the material with a silica
surface is a glass. Most preferably, the material with an affinity
to nucleic acids is a composition comprising magnetic glass
particles. The kit can further or additionally comprise a lysis
buffer containing e.g. chaotropic agents, detergents or alcohols or
mixtures thereof which allows the lysis of cells. These components
of the kit according to the invention may be provided separately in
tubes or storage containers. Depending on the nature of the
components, these may be even provided in a single tube or storage
container. The kit may further or additionally comprise a washing
solution which is suitable for the washing step of the magnetic
glass particles when DNA or RNA is bound thereto. This washing
solution may contain ethanol and/or chaotropic agents in a buffered
solution or solutions with an acidic pH without ethanol and/or
chaotropic agents as described above. Often the washing solution or
other solutions are provided as stock solutions which have to be
diluted before use. The kit may further or additionally comprise an
eluent or elution buffer, i.e. a solution or a buffer (e.g. 10 mM
Tris, 1 mM EDTA, pH 8.0) or pure water to elute the DNA or RNA
bound to the magnetic glass particles. Further, additional reagents
or buffered solutions may be present which can be used for the
purification process of a nucleic acid, i.e. DNA or RNA. A
preferred embodiment of the present invention is to use the method
or the kit of the present invention in automatable methods as e.g.
described in WO 99/16781. Automatable method means that the steps
of the method are suitable to be carried out with an apparatus or
machine capable of operating with little or no external control or
influence by a human being. Automatized method means that the steps
of the automatable method are carried out with an apparatus or
machine capable of operating with little or no external control or
influence by a human being. Only the preparation steps for the
method may have to be done by hand, e.g. the storage containers
have to filled up and put into place, the choice of the samples has
to be done by a human being and further steps known to the expert
in the field, e.g. the operation of the controlling computer. The
apparatus or machine may e.g. add automatically liquids, mix the
samples or carry out incubation steps at specific temperatures.
Typically, such a machine or apparatus is a robot controlled by a
computer which carries out a program in which the single steps and
commands are specified. Preferred automatized methods are those
which are carried out in a high-throughput format which means that
the methods and the used machine or apparatus are optimized for a
high-throughput of samples in a short time. In another embodiment
of the invention the methods or the kits according to the present
invention are used in semi-automatized process which means that
some reaction steps may have to be done manually. In a preferred
embodiment of the invention, a suspension containing MGPs according
to the present invention is taken from a storage container and
partial volumes are added to different reaction vessels. Reaction
vessels may be reaction tubes made from plastics eventually in
mictrotitreplate format contain 96 or 384 or more wells where a
reaction can be carried out. However, these vessels may be made
from other material e.g. from steel.
[0084] In preferred embodiments of the invention the kit according
to the invention is used for the purification of nucleic acids in
research, bioanalytics or diagnostics. In preferred embodiments
according to the invention the kit according to the invention or
the method according to the invention is use in a high-throughput
format, i.e. in an automatized method which allows the analysis of
a high number of different samples in a very short time.
[0085] The person skilled in the art knows from the teachings and
the example of the present invention how to identify other
proteases performing in an equivalent manner as the protease
according to the invention, i.e. the protease esperase. Thereby, it
is also possible to identify variant or mutant proteins of esperase
performing in an equivalent manner to esperase. "Mutant amino acid
sequence," "mutant protein" or "mutant polypeptide" refers to a
polypeptide having an amino acid sequence which varies from a
native sequence or is encoded by a nucleotide sequence
intentionally made variant from a native sequence. "Mutant
protein," "variant protein" or "mutein" means a protein comprising
a mutant amino acid sequence and includes polypeptides which differ
from the amino acid sequence of native esperase due to amino acid
deletions, substitutions, or both. "Native sequence" refers to an
amino acid or nucleic acid sequence which is identical to a
wild-type or native form of a gene or protein.
[0086] To find these variant or mutant proteins, he will prepare
solutions identical to the reagents and buffers described in
Example 1 whereby esperase is used as a standard for the
determination of the protease activity. Primarily, the expert in
the field will analyze the protease of interest as described in the
Chromatographic Analysis of Plasma Protein Digestion Protocol (see
Example 3). The protease in question will further be analyzed by
its properties in sample preparation with subsequent PCR
amplification and detection of the amplified product (see Example
2). Of further interest for comparison with the disclosed enzyme
esperase is the investigation of the storage stability (see Example
6) or the evaluation of the enzymatic activity in the presence of
chaotropic agents (see Example 5). Taking the results of these
investigations into account, the expert in the field can decide
whether a protease of interest performs in an equivalent manner as
the protease esperase disclosed by the present invention.
[0087] A further embodiment of the invention is an aequeous
composition of a protease according to the invention, i.e. a
protease which is at least 80% identical to the amino acid sequence
of the protease subtilisin 147 from Bacillus lentus whereby the
composition comprises 10 mM Tris acetate, 5 mM calcium chloride, 5
mM calcium acetate, 1 mM EDTA, 50% (V/V=Volume/Volume) glycerin
with a pH value of 5.5. This composition is an ideal storage buffer
for esperase (see example 6), The expert skilled in the art is able
to modify the composition of the buffer taking the teachings of
example 5 into account as long as the protease according to the
invention is equally stable in the modified buffer composition. In
a further embodiment, the amino acid sequence of the protease in
the above-desribed composition is identical to the amino acid
sequence of the protease subtilisin 147 from Bacillus lentus or the
amino acid sequence of protease is the amino acid sequence SEQ ID
NO 1, a proteolytical derivative thereof having protease activity
or the amino acid sequence SEQ ID NO 2. In another embodiment, the
amino acid sequence of the protease according to the invention in
the above-described composition is encoded by the nucleic acid
sequence SEQ ID NO 3, a part thereof or a degenerated version of
the nucleic acid sequence SEQ ID NO 3. The composition according to
the invention can be used in sample preparation or sample
preparation methods, in particular in the methods according to the
invention, for the purification of nucleic acids or in diagnostics
or diagnostical analysis.
[0088] The following examples, references, sequence listing and
figures are provided to aid the understanding of the present
invention, the true scope of which is set forth in the appended
claims. It is understood that modifications can be made in the
procedures set forth without departing from the spirit of the
invention.
EXAMPLE 1
Reagents and Buffers
[0089] 1.1 Proteases
[0090] The following proteases have been tested for their
suitability in the sample preparation process:
1 Alcalase (Novo Nordisk) Proteinase K (60 mg/ml) Roche
Diagnostics, Cat. No. 1 964 372 Subtilisin A (19 mg/ml) (Novo
Nordisk) Esperase (24 mg/ml) (Novo Nordisk) Chirazym (31 mg/ml)
Novozyme 539 (Novo Nordisk) Novo 47002 (Novo Nordisk) Novocor PL
(Novo Nordisk) Pronase (Roche Diagnostics, Cat. No. 165 921)
[0091] 1.2 Buffers:
[0092] 1.2.1 Lysis- and Binding Buffer:
[0093] Lysis-and binding buffer has been prepared from:
[0094] 5 M Guanidiumthiocyanate
[0095] 15% Polydocanol
[0096] 1% Dithiothreitol (DTT)
[0097] 15 mM Bis-TRIS, pH 6.0
[0098] 1.2.2 Washing Buffer:
[0099] Washing buffer had the following composition:
[0100] 50% Ethanol
[0101] 50 mM NaCl
[0102] 10 mM Bis-TRIS, pH 6.0
[0103] 1.2.3 Elution Buffer:
[0104] Elution buffer has been RNase-free destined water.
[0105] 1.3 Magnetic Glass Particles:
[0106] Magnetic glass particles as described in WO01/37291 have
been suspended in isopropanol at a concentration of 6 mg/ml. The
said magnetic glass particles can also be taken from the MagNA Pure
LC DNA Isolation Kit I (Roche, Mannheim, Germany).
[0107] 1.4 Buffers for the Protease Activity Assay
2 Buffer: 50 mM Tris/HCl pH 8.2 10 mM calcium chloride Substrate
solution: 200 mM Suc-Ala-Ala-Pro-Phe-p-nitroani- lide in
dimethylsulfoxide (DMSO)
[0108] 1.4 Preparation of PCR-Grade Esperase
[0109] PCR-grade esperase was prepared as follows. All equipment
was incubated overnight in 2M NaOH solution.
[0110] 1.4.1 Dialysis
[0111] 200 ml of 70 mg/ml esperase dissolved in a suitable buffer
(e.g., Esperase HP F solution from Novozyme, Copenhague) was
dialyzed over 3 days against 3.times.12 L 10 mM Tris pH 5.5, 5 mM
Calcium acetate, 5 mM calcium chloride, 1 mM EDTA, 50% glycerol.
The pH of the buffer was adjusted with acetic acid to 5.5. The
dialysate (approximately 70 ml, 150 mg protein per ml) was then
diluted with freshly prepared dialysis buffer to a protein
concentration of approximately 80 mg/ml.
[0112] 1.4.2 Ultrafiltration
[0113] Approximately. 50 ml freshly prepared dialysis buffer was
filtered through 100 kDa Omega (Pall Filtron) membrane. After that
the dialysate was filtrated through the membrane. After filtration
the membrane was washed with 20 ml dialysis buffer. The filtrate
was diluted with the filtrated dialysis buffer to a protein
concentration of 60+5 mg/ml.
[0114] 1.4.3 Sterilization
[0115] The filtrate was filtrated through a 0.22.mu. membrane (Pall
Filtron).
[0116] 1.4.4 Optional purification steps: Esperase solutions that
contained nucleases or nuclease activity, were purified by one of
the following purification steps to remove the contaminations prior
to the above described procedure:
[0117] 1.4.4.1 Ammonium Sulfate Crystallization:
[0118] 200 ml Esperase solution is diluted with 400 ml 10 mM
Tris-HCl, pH 7.5; 5 mM calcium acetate; 5 mM calcium chloride; 1 mM
EDTA. 108.6 g ammonium sulfate is added to the solution in small
portions and the solution is afterwards stirred for 1 hour at room
temperature. The solution is centrifugated. The precipitate is
washed twice with 600 ml 10 mM Tris-HCl; 5 mM calcium acetate; 5 mM
calcium chloride; 1 mM EDTA; 1.25 M ammonium sulfate pH=7.5. The
precipitate is dissolved in 200 ml 10 mM Tris-HCl; 5 mM calcium
acetate; 5 mM calcium chloride; 1 mM EDTA pH=7.5.
[0119] 1.4.4.2 Heparin-Sepharose ff-chromatography:
[0120] 4 ml Heparin-Sepharose ff. (supplier: Amershan Pharmacia) is
filled in a column and equilibrated with 10 mM Tris-HCl, pH 7.5; 5
mM Calcium acetate; 5 mM Calcium chloride; 1 mM EDTA. 200 ml
Esperase solution is passed through the column with a flow of 2
ml/min. Afterwards the column is washed with 10 ml of the
equilibration buffer. All fractions with Esperase are pooled.
EXAMPLE 2
Sample Preparation Method and Polymerase Chain Reaction
[0121] 2.1 Protease Digestion and Lysis:
[0122] 80 .mu.l protease solution is mixed with 420 .mu.l sample
material (e.g. plasma with a specific virus concentration) and
mixed. 500 .mu.l lysis- and binding buffer are added and the
solution is mixed for 10 minutes at room temperature.
[0123] 2.2 Binding:
[0124] 500 .mu.l of the suspension of magnetic glass particles in
isopropanol are added and the solution is mixed for 20 minutes at
room temperature.
[0125] 2.3 Washing:
[0126] After the binding step the magnetic glass particles are
separated from the solution by a magnet and washed five times with
750 .mu.l washing buffer per wash cycle.
[0127] 2.4 Elution:
[0128] After the last wash cycle the magnetic glass particles are
separated by a magnet from the suspension and the washing buffer is
sucked off from the magnetic glass particles and 100 .mu.l elution
buffer are added. The suspension is mixed and incubated for 15
minutes at 80.degree. C.
[0129] After the elution step the magnetic glass particles are
separated again by a magnet and the supernatant containing the
viral nucleic acid is harvested.
[0130] 2.5 Protocol Amplification/Detection:
[0131] With the exception of the primers all reagents were
purchased from Roche Molecular Biochemicals.
3 Reagent conc./PCR Master Mix HCV: Bicine Buffer (pH 8.3) 1 x
MnOAc 2.5 mM dNTP Mix with dUTP dUTP 0.6 mM dATP/dCTP/dGTP 0.2 mM
each Primer KY 80 (F) 300 nMol Primer KY 78-bio (R) 300 nMol
Tth-Polymerase 10 U Uracil-N-glycosylase (UNG) 2 U Master Mix HIV:
Bicine Buffer (pH 8.3) 1 x MnOAc 1.25 mM dNTP Mix with dUTP dUTP
0.6 mM dATP/dCTP/dGTP 0.2 mM each Primer SK 462-bio (F) 200 nMol
Primer SK 431-bio (R) 200 nMol Tth-Polymerase 15 U UNG 2 U Master
Mix HBV: DNA-Master Mix 1x MgCl.sub.2 3.0 mM Primer 1 (F) 200 nMol
Primer 2(bio (R)) 200 nMol UNG 2 U
[0132] 20 .mu.l of the eluate from the sample preparation process
which contains the target nucleic acid, e.g. viral RNA (HCV, HIV)
or viral DNA (HBV) are mixed which 100 .mu.l master mix.
Amplification is performed on a Perkin-Elmer Thermocycler 9600 with
the following thermocycler programms:
4 HCV: UNG step 1x 10 min 37.degree. C. RT step 1x 30 min
60.degree. C. 1x 1 min 95.degree. C. PCR 2x 10 sec 95.degree. C. 20
sec 60.degree. C. 33x 15 sec 90.degree. C. 20 sec 60.degree. C. 1x
7 min 72.degree. C. HIV: UNG step 1x 10 min 37.degree. C. RT step
1x 30 min 60.degree. C. PCR 4x 10 sec 95.degree. C. 10 sec
55.degree. C. 10 sec 72.degree. C. 31x 10 sec 90.degree. C. 10 sec
60.degree. C. 10 sec 72.degree. C. HBV: UNG step 1x 10 min
37.degree. C. PCR 35x 30 sec 92.degree. C. 30 sec 55.degree. C. 40
sec 72.degree. C.
[0133] For the detection of the amplified material, a very
sensitive nonisotopic approach based on electrochemiluminescence
(ECL) was used. Ruthenium-tris(bipyridyl)-labeled oligonucleotides
(capture probes) were hybridized specifically to the biotinylated
denatured amplicons. Subsequent, this hybrid was bound to the
surface of streptavidin-coated magnetic beads. After the beads were
captured on an electrode by using a permanent magnet, the ECL
reaction of the ruthenium label was triggered by voltage
application. For details of the ECL detection process, see Hoyle et
al. (13). The totally automated ECL detection was performed on an
instrumental platform (preprototype of Elecsys 1010; Boehringer
Mannheim GmbH).
5 HCV: KY80: SEQ ID NO: 4 KY78: SEQ ID NO: 5 Probe: SEQ ID NO: 6
HIV: SK 462: SEQ ID NO: 7 SK 431: SEQ ID NO: 8 Probe: SEQ ID NO: 9
HBV: Primer 1: SEQ ID NO: 10 Primer 2: SEQ ID NO: 11 Probe: SEQ ID
NO: 12
[0134]
6 Result Proteinase K Pronase Subtilisin A Esperase Chirazym (ECL
(ECL (ECL (ECL (ECL counts .times. counts .times. counts .times.
counts .times. counts .times. Virus 10.sup.-3) 10.sup.-3)
10.sup.-3) 10.sup.-3) 10.sup.-3) HIV 278 62 62 210 214 HCV 184 22
49 179 249 HBV 371 30 241 300 446
[0135] Only the use of esperase and chirazym for the degradation of
plasma proteins in the sample preparation process results in an ECL
signal comparable to the signal generated by the use of proteinase
K in the sample preparation process.
EXAMPLE 3
Protocol Chromatographic Analysis of Plasma Protein Digestion
[0136] Protein digestion and lysis were carried out as described.
Each 100 .mu.l of the lysated solution were injected onto an high
pressure liquid chromatography instrument (HPLC) (Dionex,
Gynkothek) and separated on an reversed phase column (C4, Vydac,
4.6 mm.times.150 mm) in a linear gradient of 0-80% acetonitrile in
0.1% trifluoroacetic acid (TFA). Peaks were detected at a
wavelength of 220 nm and 280 nm.
7 Plasma Protein Digestion with Protease stressed by thermal
treatment (after 3 day incubation at 45.degree. C. Protease With
unstressed Protease in storage buffer* Esperase ++ ++ Proteinase K
++ ++ Pronase ++ - Subtilisin A ++/+ + Alcalase + not tested
Novozyme 539 + not tested Novo 47002 - not tested Novocor PL - not
tested *Storage buffer composition: 10 mM Tris acetate, 5 mM
calcium chloride, 5 mM calcium acetate, 1 mM EDTA, 50% (V/V)
glycerin with a pH value of 5.5
[0137] In FIG. 1, the comparison of the digestion of EDTA plasma
versus citrate plasma with Esperase (see FIG. 1a) and proteinase K
(see FIG. 1b) is shown.
EXAMPLE 4
Evaluation of the pH Optimum
[0138] The pH optimum of esperase was compared to the pH optimum of
proteinase K using the buffers as basically described under 1.1.4
with a varying pH. The pH optimum was more in the neutral pH region
as compared to proteinase K (see FIG. 2).
[0139] Sample: 10 mg protein are dissolved in 1 ml distilled water.
Before the determination, the sample is diluted with dest. water so
that the increase in the extinction in the test is between 0.02 and
0.05 E.
[0140] Sample Buffer:
[0141] pH-range: 5.5 bis 7.5: 50 mM Bis-Tris+10 mM CaCl.sub.2 are
adjusted with 2 N HCl or 2 N NaOH to the respective pH.
[0142] pH-range 7.5 bis 9.5: 50 mM Tris-Base+10 mM CaCl.sub.2 are
adjusted with 2 N HCl or 2 N NaOH to the respective pH.
[0143] Substrate: Suc-Ala-Ala-Pro-Phe-p-nitroanilide (200 mM
dissolved in Dimethyl sulfoxide (DMSO)).
[0144] Measurement:
[0145] Pipetting scheme: 2.00 ml sample buffer
[0146] 0.2 ml substrate
[0147] 0.05 ml sample
[0148] Temperature for measurement: 25.degree. C.
[0149] Wavelength for measurement: 405 nm
[0150] Evaluation: The linear increase in extinction (de/min) is
determined between 2 and 6 ml.
[0151] Layer thickness: 1 cm 1 Activity = 2.07 * dE / min 10.4 ( )
* 0.05 * 1 * dilution ( U / ml )
[0152] Relative Activity: For each sample, the highest measured
activity is regarded as the value of 100% and the activities at
other pH-values are evaluated by determining the percental relation
to this value.
EXAMPLE 5
Evaluation of the Enzymatic Activity in the Presence of Chaotropic
Agents
[0153] The enzymatic activity of esperase was compared to the
enzymatic activity of proteinase K in the presence of chaotropic
agents using the buffers as basically described under 1.1.4 with
increasing amounts of chaotropic agent. Esperase retained more
activity in the presence of chaotropic agents (see FIG. 3 and FIG.
4). This lower residual activity is advantageous as the protein
digestion by esperase is very quick in the presence of chaotropic
agent (.ltoreq.1 ml) and as esperase has a low residual activity.
This is of advantage as less active esperase is transferred into
the amplification reaction where it may disturb the amplification
reaction.
[0154] Protease solution: 20 mg/ml Protease
[0155] Sample: 500 .mu.l chaotropic agent
[0156] 50 .mu.l protease solution.
[0157] The activity of the protease is determined in various
solutions. Then, the sample is incubated for 15 min at 25.degree.
C. and the residual activity determined in various agents.
[0158] Determination of the Activity:
[0159] Test buffer: 50 mM Tris.HCl pH=8.2; 10 mM CaCl.sub.2
[0160] Substrate: 200 mM Suc-Ala-Ala-Prp-Phe-p-nitroanilide in
DMSO
[0161] Measuring temperature: 25.degree. C.
[0162] Measuring wavelength: 405 nm
[0163] Evaluation: see evaluation of the pH Optimum.
Example 6
Storage Stability
[0164] The stability of the proteases was determined by following
the proteolytic activity under thermal stress in storage buffer
(composition: 10 mM Tris acetate, 5 mM calcium chloride, 5 mM
calcium acetate, 1 mM EDTA, 50% (V/V) Glycerin with a pH value of
5.5). A kinetic assay with Suc-Ala-Ala-Pro-Phe-p-nitroanilide as a
substrate was used. Shortly before use the protease sample has to
be diluted to a concentration of 1-3 .mu.g/ml with distilled water.
2 ml buffer were mixed with 0.02 ml substrate and 0.05 ml diluted
sample. The release of p-nitroaniline from the substrate at
25.degree. C. was measured photometrically at 405 nm. The
time-curve of the stability of Esperase in comparison to proteinase
K is shown in FIG. 5. The result of this experiment is that it
could be shown that Esperase is very stable in storage buffer even
after a prolonged period of time.
8 Remaining activity after 3 day incubation at 45.degree. C.
Protease in storage buffer (composition see above) Esperase 88%
Proteinase K 94% Pronase 89% Subtilisin A 45%
List of References
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[0166] Abramson and Myers, 1993, Current Opinion in Biotechnology
4:41-47
[0167] Anal. Biochem. 121, 382-387 (1982)
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[0169] Anal. Biochem. 201, 166-169 (1992)
[0170] Ausubel et al.: Current Protocols in Molecular Biology 1987,
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[0171] Barany, 1991, PCR Methods and Applic. 1:5-16
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[0173] DE 3724442
[0174] EP 110165
[0175] EP 396 608
[0176] EP 439 182
[0177] GB 91/00212
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patents are incorporated herein by reference.
Sequence CWU 1
1
12 1 361 PRT Bacillus lentus 1 Met Arg Gln Ser Leu Lys Val Met Val
Leu Ser Thr Val Ala Leu Leu 1 5 10 15 Phe Met Ala Asn Pro Ala Ala
Ala Gly Gly Glu Lys Lys Glu Tyr Leu 20 25 30 Ile Val Val Glu Pro
Glu Glu Val Ser Ala Gln Ser Val Glu Glu Ser 35 40 45 Tyr Asp Val
Asp Val Ile His Glu Phe Glu Glu Ile Pro Val Ile His 50 55 60 Ala
Glu Leu Thr Lys Lys Glu Leu Lys Lys Leu Lys Lys Asp Pro Asn 65 70
75 80 Val Lys Ala Ile Glu Glu Asn Ala Glu Val Thr Ile Ser Gln Thr
Val 85 90 95 Pro Trp Gly Ile Ser Phe Ile Asn Thr Gln Gln Ala His
Asn Arg Gly 100 105 110 Ile Phe Gly Asn Gly Ala Arg Val Ala Val Leu
Asp Thr Gly Ile Ala 115 120 125 Ser His Pro Asp Leu Arg Ile Ala Gly
Gly Ala Ser Phe Ile Ser Ser 130 135 140 Glu Pro Ser Tyr His Asp Asn
Asn Gly His Gly Thr His Val Ala Gly 145 150 155 160 Thr Ile Ala Ala
Leu Asn Asn Ser Ile Gly Val Leu Gly Val Arg Pro 165 170 175 Ser Ala
Asp Leu Tyr Ala Leu Lys Val Leu Asp Arg Asn Gly Ser Gly 180 185 190
Ser Leu Ala Ser Val Ala Gln Gly Ile Glu Trp Ala Ile Asn Asn Asn 195
200 205 Met His Ile Ile Asn Met Ser Leu Gly Ser Thr Ser Gly Ser Ser
Thr 210 215 220 Leu Glu Leu Ala Val Asn Arg Ala Asn Asn Ala Gly Ile
Leu Leu Val 225 230 235 240 Gly Ala Ala Gly Asn Thr Gly Arg Gln Gly
Val Asn Tyr Pro Ala Arg 245 250 255 Tyr Ser Gly Val Met Ala Val Ala
Ala Val Asp Gln Asn Gly Gln Arg 260 265 270 Ala Ser Phe Ser Thr Tyr
Gly Pro Glu Ile Glu Ile Ser Ala Pro Gly 275 280 285 Val Asn Val Asn
Ser Thr Tyr Thr Gly Asn Arg Tyr Val Ser Leu Ser 290 295 300 Gly Thr
Ser Met Ala Thr Pro His Val Ala Gly Val Ala Ala Leu Val 305 310 315
320 Lys Ser Arg Tyr Pro Ser Tyr Thr Asn Asn Gln Ile Arg Gln Arg Ile
325 330 335 Asn Gln Thr Ala Thr Tyr Leu Gly Ser Pro Ser Leu Tyr Gly
Asn Gly 340 345 350 Leu Val His Ala Gly Arg Ala Thr Gln 355 360 2
268 PRT Bacillus lentus 2 Gln Thr Val Pro Trp Gly Ile Ser Phe Ile
Asn Thr Gln Gln Ala His 1 5 10 15 Asn Arg Gly Ile Phe Gly Asn Gly
Ala Arg Val Ala Val Leu Asp Thr 20 25 30 Gly Ile Ala Ser His Pro
Asp Leu Arg Ile Ala Gly Gly Ala Ser Phe 35 40 45 Ile Ser Ser Glu
Pro Ser Tyr His Asp Asn Asn Gly His Gly Thr His 50 55 60 Val Ala
Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly 65 70 75 80
Val Arg Pro Ser Ala Asp Leu Tyr Ala Leu Lys Val Leu Asp Arg Asn 85
90 95 Gly Ser Gly Ser Leu Ala Ser Val Ala Gln Gly Ile Glu Trp Ala
Ile 100 105 110 Asn Asn Asn Met His Ile Ile Asn Met Ser Leu Gly Ser
Thr Ser Gly 115 120 125 Ser Ser Thr Leu Glu Leu Ala Val Asn Arg Ala
Asn Asn Ala Gly Ile 130 135 140 Leu Leu Val Gly Ala Ala Gly Asn Thr
Gly Arg Gln Gly Val Asn Tyr 145 150 155 160 Pro Ala Arg Tyr Ser Gly
Val Met Ala Val Ala Ala Val Asp Gln Asn 165 170 175 Gly Gln Arg Ala
Ser Phe Ser Thr Tyr Gly Pro Glu Ile Glu Ile Ser 180 185 190 Ala Pro
Gly Val Asn Val Asn Ser Thr Tyr Thr Gly Asn Arg Tyr Val 195 200 205
Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val Ala 210
215 220 Ala Leu Val Lys Ser Arg Tyr Pro Ser Tyr Thr Asn Asn Gln Ile
Arg 225 230 235 240 Gln Arg Ile Asn Gln Thr Ala Thr Tyr Leu Gly Ser
Pro Ser Leu Tyr 245 250 255 Gly Asn Gly Leu Val His Ala Gly Arg Ala
Thr Gln 260 265 3 1086 DNA Bacillus lentus 3 atgagacaaa gtctaaaagt
tatggttttg tcaacagtgg cattgctttt catggcaaac 60 ccagcagcag
caggcgggga gaaaaaggaa tatttgattg tcgtcgaacc tgaagaagtt 120
tctgctcaga gtgtcgaaga aagttatgat gtggacgtca tccatgaatt tgaagagatt
180 ccagtcattc atgcagaact aactaaaaaa gaattgaaaa aattaaagaa
agatccgaac 240 gtaaaagcca tcgaagagaa tgcagaagta accatcagtc
aaacggttcc ttggggaatt 300 tcattcatta atacgcagca agcgcacaac
cgcggtattt ttggtaacgg tgctcgagtc 360 gctgtccttg atacaggaat
tgcttcacac ccagacttac gaattgcagg gggagcgagc 420 tttatttcaa
gcgagccttc ctatcatgac aataacggac acggaactca cgtggctggt 480
acaatcgctg cgttaaacaa ttcaatcggt gtgcttggtg tacgaccatc ggctgacttg
540 tacgctctca aagttcttga tcggaatgga agtggttcgc ttgcttctgt
agctcaagga 600 atcgaatggg caattaacaa caacatgcac attattaata
tgagccttgg aagcacgagt 660 ggttctagca cgttagagtt agctgtcaac
cgagcaaaca atgctggtat tctcttagta 720 ggggcagcag gtaatacggg
tagacaagga gttaactatc ctgctagata ctctggtgtt 780 atggcggttg
cagcagttga tcaaaatggt caacgcgcaa gcttctctac gtatggccca 840
gaaattgaaa tttctgcacc tggtgtcaac gtaaacagca cgtacacagg caatcgttac
900 gtatcgcttt ctggaacatc tatggcaaca ccacacgttg ctggagttgc
tgcacttgtg 960 aagagcagat atcctagcta tacgaacaac caaattcgcc
agcgtattaa tcaaacagca 1020 acgtatctag gttctcctag cctttatggc
aatggattag tacatgctgg acgtgcaaca 1080 caataa 1086 4 24 DNA
Hepatitis C virus 4 gcagaaagcg tctagccatg gcgt 24 5 24 DNA
Hepatitis C virus modified_base (1) Biotin derivatization 5
ctcgcaagca ccctatcagg cagt 24 6 21 DNA Hepatitis C virus
modified_base (1) Ruthenium3+-(tris-bipyridyl)-derivatisation 6
gtcgtgcagc ctccaggacc c 21 7 30 DNA Human immunodeficiency virus
modified_base (1) Biotin derivatization 7 agttggagga catcaagcag
ccatgcaaat 30 8 27 DNA Human immunodeficiency virus modified_base
(1) Biotin derivatization 8 tgctatgtca gttccccttg gttctct 27 9 20
DNA Human immunodeficiency virus modified_base (1)
Ruthenium3+-(tris-bipyridyl)-derivatisation 9 atcaatgagg aagctgcaga
20 10 18 DNA Hepatitis B virus 10 ggagtgtgga ttcgcact 18 11 18 DNA
Hepatitis B virus modified_base (1) Biotin derivatization 11
tgagatcttc tgcgacgc 18 12 18 DNA Hepatitis B virus modified_base
(1) Ruthenium3+-(tris-bipyri- dyl)-derivatisation 12 agaccaccaa
atgcccct 18
* * * * *